Enzyme detection biosensors

ABSTRACT

The present invention provides a biosensor for use in detecting the presence of an enzyme or enzymes in a sample. The biosensor comprises a membrane and means for determining the impedance of the membrane. The membrane includes ionophores therein to which are attached linkers. The linkers are cleavable by the enzyme or enzymes to be detected, with the cleavage of the linker causing a change in the ability of ions to pass through the membrane via the ionophores.

This application claims benefit to Provisional Application No.60/011,314 filed Feb. 8, 1996.

The present invention relates to biosensors and methods involving theuse of these biosensors in detecting the presence of enzymes bydetecting their enzymatic activity.

BACKGROUND OF THE INVENTION

A number of proteins which are useful as immunodiagnostic analytes anddisease markers have the additional property of enzymatic activity, inparticular protease activity. In addition, other classes of proteinsexhihibit nuclease activity.

Prostate Specific Antigen (PSA), a diagnostic marker for prostate canceris an example of a protein which exhibits protease activity, and belongsto the class of proteins known as the serine proteases. Examples ofother proteases which are important immunodiagnostic markers includeblood coagulation enzymes, elastase, cathepsin B.

There are also a number of important industrial enzymes such assubtilisin, papain and α-amylase.

Examples of important nucleases are restriction enzymes, e.g., BamH1.Hind III, polymerases which can act as nucleases under certainconditions. e.g., T4 DNA polymerase, reverse transcriptase, which actsas an Rnase under certain conditions, e.g. Rnase H, and exo- andendo-nucleases, e.g. S1 nuclease.

Current diagnostic tests employ immunoassays for the detection of PSA(e.g. a number of analytical instruments such as Abbott's AXsym.Boehringer Mannheim's Elecsys, and CIBA-Corning's ACS-180. all haveELISA-based PSA tests). These tests use antibodies raised against thePSA molecule which recognise the specific epitope sites within theprotein molecule.

A variation on these approaches is disclosed in International Patentapplication No. PCT/AU95/00536. In this reference there is disclosed arange of substrates specifically cleaved by PSA. There is alsodisclosure in this reference of an assay system for proteases such asPSA which make use of the activity of the protease. This assay systeminvolves the use of a ligand to capture the PSA and the subsequent useof a substrate for the PSA.

SUMMARY OF THE INVENTION

The present inventors have developed devices and methods for thedetection of enzymes which make use of the protein's protease activity.These devices and methods involve the use of membrane based biosensors.Information regarding such biosensors can be found in InternationalPatent Application Nos PCT/AU88/00273, PCT/AU89/00352, PCT/AU90/00025,PCT/AU92/00132, PCT/AU93/00509, PCT/AU93/00620, PCT/AU94/00202 andPCT/AU95/00763. The disclosure of each of these applications is includedherein by reference.

The present invention involves providing a substrate for the enzyme tobe detected and then sensing the digestion of the substrate by theenzyme. This may be achieved in a number of ways, for example thedigestion of the substrate may remove a group from the ionophore therebyreleasing the ionophore so that it diffuses laterally within themembrane or may result in an increase in the ability of ions to passthrough the ionophore simply by a reduction in “steric” hindrance.Alternatively the digestion of the substrate when attached to a membranespanning component may result in the release of the ionophore such thatit may diffuse laterally within the membrane Clearly this could also beachieved by digestion of substrates attached to both the ionophore andmembrane spanning component.

In another arrangement the digestion of the substrate results in therelease of ionophore including probe which then inserts itself into themembrane.

Accordingly, in a first aspect the present invention consists in abiosensor for use in detecting the presence of an enzyme in a sample,the biosensor comprising a membrane and means for determining theimpedance of the membrane, the membrane having ionophores therein towhich are attached linkers, the linkers being cleavable by the enzyme tobe detected, the cleavage of the linker causing a change in the abilityof ions to pass through the membrane via the ionophores.

In a preferred embodiment of the present invention the linker isattached to the membrane such that the ionophore is prevented fromdiffusing laterally within the membrane. It is preferred that the linkeris attached to membrane spanning components provided in the membrane.This attachment may be achieved in a number of ways such as covalentattachment, however, it is presently preferred that the attachment isachieved by providing on each of the linker and membrane spanningcomponent one member of a ligand binding pair. A preferred ligandbinding pair is biotin streptavidin. In another preferred arrangementboth the membrane spanning component and the linker are provided withmoieties which are both bound to the same molecule, for example biotinis provided on both the membrane spanning component and the linker andthere is cross-linking via streptavidin.

The moiety on the membrane spanning component may also be attached via alinker. This may be the same linker as that provided on the ionophore ormay be different.

In a further preferred embodiment the membrane comprises a first andsecond layer of a closely packed array of amphiphilic molecules, aplurality of ionophores and a plurality of membrane-spanning lipidsprevented from lateral diffusion in the membrane, the ionophorescomprising first and second half membrane spanning monomers, the firsthalf membrane spanning monomers being provided in the first layer andthe second half membrane spanning monomers being provided in the secondlayer, the first half membrane spanning monomers being prevented fromlateral diffusion in the first layer, the second half membrane spanningmonomers being linked to the membrane spanning lipids via the linker.Following cleavage of the linker by the enzyme the second half membranespanning monomers can diffuse laterally within the second layerindependent of the first half membrane spanning monomers.

In a second aspect the present invention consists in a biosensor for usein detecting the presence of an enzyme in a sample, the biosensorcomprising a membrane and means for determining the impedance of themembrane, the membrane having a plurality of ionophores and a pluralityof membrane-spanning components therein, the membrane-spanningcomponents having attached thereto linker molecules to which areconnected the ionophores, the linker molecules being cleavable by theenzyme to be detected, the cleavage of the linker molecules causing achange in the ability of ions to pass through the membrane via theionophores.

In a preferred embodiment the membrane comprises a first and secondlayer of a closely packed array of amphiphilic molecules and themembrane-spanning components are prevented from lateral diffusion in themembrane. Preferably the ioniophores comprise first and second halfmembrane spanning monomers, the first half membrane spanning monomersbeing provided in the first layer and the second half membrane spanningmonomers being provided in the second layer with the first half membranespanning monomers being prevented from lateral diffusion in the firstlayer. The second half membrane spanning monomers are connected to themembrane-spanning components via the linker molecule.

The ionophores in both these aspects are preferably gramicidin oranalogues thereof.

While a range of enzymes can be detected using the biosensor or thepresent invention the biosensor is particularly useful in the detectionof proteases, in particular those of clinical importance such as PSA,fibrinogen etc.

In a third aspect the present invention consists in a biosensor for thedetection of enzymes comprising first and second zones, means to allowaddition of a sample suspected to contain an enzyme to the first zone,the first zone containing a probe linked to a carrier via a linkercleavable by the enzyme and means to allow passage of unlinked probefrom the first zone to the second zone; the second zone including amembrane the impedance of which is dependent on the presence or absenceof the probe and means to measure the impedance of the membrane.

In a preferred embodiment of this aspect of the present invention themembrane comprises a first and a second layer of a closely packed arrayof amphiphilic molecules and a plurality of ionophores comprising afirst and second half membrane spanning monomers, the first halfmembrane spanning monomers being provided in the first layer and thesecond half membrane spanning monomers being provided in the secondlayer. The second half membrane spanning monomers being capable oflateral diffusion within the second layer independent of the first halfmembrane spanning monomers, the first half membrane spanning monomersbeing prevented from lateral diffusion in the first layer, and a ligandprovided on at least the second half membrane spanning monomers, saidligand being reactive with the probe or a portion thereof, the bindingof the probe to the ligand causing a change in the relationship betweenthe first half membrane spanning monomers and the second half membranespanning monomers such that the flow of ions across the membrane via theionophores is allowed or prevented.

In a preferred embodiment the probe includes streptavidin and the ligandincludes biotin.

In yet another preferred embodiment the probe includes an ionophore suchthat when the probe comes into contact with the membrane the ionophoreinserts itself into the membrane changing the impedance of the membrane,As an example of such an arrangement the probe may include valinomycinwhich inserts itself into the membrane.

In a preferred embodiment of the present invention the enzyme to bedetected is a protease in particular Prostate Specific Antigen. In thiscase it is preferred that the linker or linker molecule includes thesequence Ala-Val-Tyr.

As will be recognised by those skilled in the art the actual linker usedwill depend on the enzyme to be detected. Examples of some enzymes andtheir corresponding substrates are set out in Whittaker et al.Analytical Biochemistry: 220, 238-243 (1994), the disclosure of which isincorporated by cross-reference.

In a further aspect the present invention consists in a method ofdetecting the presence of an enzyme in a sample comprising adding thesample to the biosensor of the first or second or third aspect of thepresent invention and measuring the change in impedance of the membrane.

As will be readily apparent the biosensors and methods of the presentinvention do not detect total enzyme: they detect only active enzyme.This is important as in a number of situations it is the amount ofactive enzyme present which is of importance not simply the total amountof enzyme present as would be measured in a standard sandwich ELISA.

It will also be apparent that the sensors of the present invention canbe used to detect a wide range of enzymes. These enzymes includenucleases, protease amylases etc. The sensors are adapted to theparticular enzyme to be detected by adjusting the make-up of the linker.For example to detect proteases the linker will typically include apeptide portion which is cleaved by the enzyme. Information regardingpeptide sequences cleaved by specific proteases is provided in Whittakeret al referred to above, Where the enzyme to be detected is a nucleasethe linker will typically include a nucleic acid sequence. Informationregarding specific sequences cleaved by specific enzymes can be found in“Current Protocols in Molecular Biology” Ausebel et al (1987) John Wiley& Sons, N.Y.

The sensors of the present invention may also find use in drugdevelopment for determining DNA-drug binding sites. The sensors couldalso be used in determining DNA-protein binding sites. The sensors mayalso find use in diagnosing infection. For example the sensors could beused to detect enzyme activity specifically associated with a pathogen.

Industrially and clinically relevant proteases and substrates includethrombin and serine proteases including PSA. A list of lysis enzymes isfound in “Specificity of Proteolysis” Borivoj Keil (1992) SpringerVerlag N.Y. pp. 283-323. Useful ones are the serine and cysteineproteases. See also “Proteolytic Enzymes”: a Practical Approach” R. J.Benyon & J. S. Bond (eds) 1989 Oxford University Press N.Y. p232, pp.241-249. Commercially significant proteases and protease inhibitors forwhich the present technology is relevant are available in serine,cysteine, aspartic and metallo types. The serine proteases include theendoproteinase-Arg-C, -Glu-C, Lys-C, factor Xa, proteinase K. subtilisinand trypsin, and the exopeptidases acylamino-acid-releasing enzyme,carboxypeptidase P, and carboxypeptidase Y. The cysteine proteasesinclude the endopeptidases bromelain, cathepsin B. clostripain, papain,and the exopeptidases cathepsin C and pyroglutamate aminopeptidase. Theaspartic proteases include the endopeptidases cathepsin D and pepsin.The metallo proteases include the endopeptidase thermolysin and theexopeptidases aminopeptidase M, carboxypeptidase-A, -B and leucineaminopeptidase. The listing is not intended to be exclusive andindicates the broad utility of the present invention. Other commerciallyuseful proteases are listed in the publications cited above, which areincluded herein by reference. For example it also includes theendopeptide endoproteinase-Asp-N of unknown type.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the nature of the present invention may be more clearlyunderstood preferred forms thereof will now be described by reference tothe following Examples and accompanying Figures, in which:

FIG. 1 is a schematic representation of an embodiment of the device ofthe third aspect of the present invention;

FIG. 2 shows an embodiment of the first and/or second aspect of theinvention;

FIG. 3 is Gramicidin B;

FIG. 4 is Spanner Lipid;

FIG. 5 is MAAD;

FIG. 6 is Lipid A′

FIG. 7 is DPE-PC;

FIG. 8 is GDPE;

FIG. 9 is biotinylated gramicidin E;

FIG. 10 is a plot of admittance at minimum phase with respect to bindingof streptavidin to biotinylated gramicidin E;

FIG. 11 is a plot of admittance at minimum phase with respect to sensormembranes to which streptavidin was bound;

FIG. 12 is membrane spanner lipid C;

FIG. 13 is a plot of admittance at minimum phase with respect to bindingof DNA target I to sensor wells;

FIG. 14 is a plot of admittance at minimum phase with respect toaddition of DNase 1 for sensor wells;

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

FIG. 1 shows a schematic representation of an embodiment of the deviceof the third aspect of the present invention. As can be seen from thisFigure the device 10 includes a first zone 11 and a second zone 12.First zone 11 is provided with polymer beads 13 (carrier) linked tostreptavidin 14 (probe) via a peptide linker 15. The peptide linker 15is cleavable by the protease 16.

As shown in this Figure upon addition of the protease (or a nuclease) 16the streptavidin 14 is released and passes to the second zone 12. Secondzone 12 includes a biosensor membrane 17 which detects the presence ofstreptavidin 14. Streptavidin 14 reaching biosensor membrane 17 causes achange in the impedance of the membrane.

FIG. 2 shows an embodiment of the first and/or second aspect of theinvention. As shown in FIG. 2 the biosensor membrane 20 includes amembrane 21 and eletrode 22. The membrane 21 has a first layer 23 andsecond layer 24 of arrays of amphiphilic molecules. Included in layer 24is a first half membrane-spanning monomer 25 which is prevented fromlateral diffusion within the membrane. Layer 23 includes a second halfmembrane-spanning monomer 26. The membrane also includes amembrane-spanning lipid 27 which is also prevented from diffusinglaterally within the membrane. The second half membrane-spanning monomer26 is linked to the membrane-spanning lipid 27 via a peptide 28. Thepeptide 28 is cleavable by protease 29. Upon cleavage of the peptide 28by protease 29 the half membrane-spanning monomer 26 is free to diffuselaterally within the membrane. This results in a change in impedance ofthe membrane.

EXAMPLES Example 1

Protease cleavage of streptavidin-gramicidin linkage

1st layer: 9.3 nM Linker Gramicidin B (FIG. 3) 1.1 μM Membrane SpannerLipid D (FIG. 4) 37 μM MAAD (FIG. 5) 75 μM Linker Lipid A (FIG. 6)

2nd layer: 10 mM (DPE-PC (FIG. 7):GDPE (FIG. 8)=7:3): BiotinylatedGramicidin E (FIG. 9)=66,677:1 in ethanol.

Electrodes with freshly evaporated gold (1000 Å) on a chrome adhesionlayer (200 Å on glass microscope slides) were dipped into an ethanolicsolution of the first layer components for 1 hour at room temperature,rinsed with ethanol, then stored at 4° C. under ethanol until used forimpedance measurements. The slide was clamped into a block containingteflon coated wells which defined the area of the working electrode asapproximately 16 mm².

5 μL of the second layer was added to the working electrode beforeaddition of a 150 μL volume of phosphate buffered saline (6.26 mM NaCl,59.4 mM NaH₂PO₄. 2H₂O, 2.53 mM Na₂HPO₄. 12H₂O, 50 mM EDTA at pH 7.4;PBS). The electrode was then washed 4 times using PBS and raised to 60°C. over a 30 minute period. Streptavidin was added to the sensor wells(5 μL 0.01 mg/ml in PBS) and incubated. The binding of streptavidin tothe biotinylated gramicidin E gave a decrease in the admittance atminimum phase (FIG. 10). After 15 minutes the excess streptavidin waswashed out with PBS. Wells with no added streptavidin were run ascontrols.

Proteinase K was added to sensing and control wells to give end wellconcentration at 12.5 mg/ml (Boehringer Mannheim D-68298 made in PBS).Addition of Proteinase K to control wells caused no significant changein membrane admittance characteristic. Sensor membranes to whichstreptavidin was bound exhibited an increase in admittance at minimumphase (FIG. 11). The amount and rate of increase of admittance atminimum phase is related to the amount of proteinase K present in thetest solution and therefore can be used to determine enzymatic activityin test solutions.

Example 2

Dnase 1 cleavage of DNA-bound channels

1st layer: 9.3 nM Linker Gramicidin B 1.1 μM Membrane Spanner Lipid D27.5 nM Membrane Spanner Lipid C (FIG. 12) 37 μM MAAD 75 μM Linker LipidA

2nd layer: 14 mM (DPE-PC:GDPE=7:3): Biotinylated Gramicidin E=50.000:1in ethanol.

Electrodes with freshly evaporated gold (1000 Å) on a chrome adhesionlayer (200 Å) on glass microscope slides) were dipped into an ethanolicsolution of the first layer components for 1 hour at room temperature.rinsed with ethanol. then stored at 4° C. under ethanol until used forimpedance measurements. The slide was clamped into a block containingteflon coated wells which defined the area of the working electrode asapproximately 16 mm².

5 μL of the second layer was added to the working electrode beforeaddition of a 180 μL volume of phosphate buffered saline (10 mM NaH₂PO₄,1 mM KH₂PO₄, 137 mM NaCl, 2.7 mM KCl: PBS). The electrode was washed 4times using PBS. These steps were carried out at room temperature. Allthe subsequent steps were carried out at 30° C. Streptavidin was addedto all the wells (5 μL 0.01 mg/ml in PBS) and allowed to react withbiotinylated gramicidin E for 10-15 minutes before washing out excessunbound streptavidin with PBS, 5 μL of a 1:1 mixture of DNA probe F (200nM): DNA probe G (200 nM in PBS) was added to the sensor wells. A DNAnon-specific binding probe H (5 μL 400 nM in PBS) was added to controlwells. Binding probe H is non-complementary to the target DNA ofinterest and hence target DNA should not bind. The probes were allowedto react with streptavidin for 10-15 minutes then excess unbound probeswere washed out with PBS. 100 μL of DNA target I (10 nM) in PBS wasadded to each well. The binding of DNA target I to the sensor wells gavea decrease in the admittance at minimum phase, but no significant changein membrane admittance in control wells (FIG. 13). After 15 minutesunbound DNA target I was washed out with DNase 1 activation buffer.DNase 1 activation buffer consists of 50 nM Tris. HCl, pH 7.6. 50 nMNaCl, 10 nM MgCl₂, 10 nM MnCl₂, 0.2 mg/mL BSA. DNase 1 was added (2 μL 1mg/mL in a 50% w/v glycerol solution of 20 mM Tris.HCl, pH 7.6. 1 mMMgCl₂) to sensor and control wells. Addition of DNase 1 gave an increasein admittance at minimum phase for sensor wells. but no significantchange for control wells (FIG. 14). The amount and rate of increase ofadmittance at minimum phase is related to the amount of DNase 1 presentin the test solution and therefore can be used to determine enzymaticactivity in test solutions.

DNA probe F:

5′biotinylated listeria probe DNA with a 31-atom phosphoramidite linkergroup between the biotin and DNA.

5′-bio-L-M-ATAGTTTTATGGGATTAGC-3′

DNA probe G:

5′biotinylated cholera toxin probe DNA with a 13-atom phosphoramiditelinker group between the biotin and DNA.

5′-bio-L-CTCCGGAGCATAGAGCTTGGAGG-3′

DNA non-specific binding probe H:

5′biotinvlated 15-mer oligonucleotide with a 31-atom phosphoramiditelinker group between the biotin and DNA, which is non-complementary toall parts of the target DNA sequence.

5′-bio-L-M-ATTGCTACGTATACG-3′

DNA target I:

52 base DNA sequence containing the 19-base listeria sequence, a 10 base‘spacer’ and the 23 base cholera toxin sequence.

5′-GCTAATCCCATAAAACTATGCATGCATATCCTCCAAGCTCTATGCTCCGGAG-3′

where:

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are.therefore. to be considered in all respects as illustrative and notrestrictive.

4 1 23 DNA Artificial Sequence Description of Artificial Sequencecholeratoxin probe 1 ctccggagca tagagcttgg agg 23 2 15 DNA Artificial SequenceDescription of Artificial Sequencenon-specific binding probe 2attgctacgt atacg 15 3 19 DNA Artificial Sequence Description ofArtificial Sequencelisteria probe 3 atagttttat gggattagc 19 4 52 DNAArtificial Sequence Description of Artificial Sequence5′, 19 baselisteria sequence, 10 base spacer, 23 base cholera toxin sequence 4gctaatccca taaaactatg catgcatatc ctccaagctc tatgctccgg ag 52

What is claimed is:
 1. A biosensor for use in detecting the presence ofan enzyme or enzymes in a sample, the biosensor comprising a membraneand means for determining the impedance of the membrane positionedrelative to the membrane such that the impedance of the membrane may bedetermined, the membrane having ionophores therein to which are attachedlinkers, the linkers being cleavable by the enzyme or enzymes to bedetected, the cleavage of the linker causing a change in the ability ofions to pass through the membrane via the ionophores.
 2. A biosensor asclaimed in claim 1 in which the linker is attached to the membrane suchthat the ionophore is prevented from diffusing laterally within themembrane.
 3. A biosensor as claimed in claim 2 in which the linker isattached to membrane spanning components provided in the membrane.
 4. Abiosensor as claimed in claim 3 in which the linker is attached to themembrane spanning component via a ligand binding pair.
 5. A biosensor asclaimed in claim 1 in which the membrane comprises a first and secondlayer of a closely packed array of amphiphilic molecules, a plurality ofionophores and a plurality of membrane-spanning lipids prevented fromlateral diffusion in the membrane, the ionophores comprising first andsecond half membrane spanning monomers, the first half membrane spanningmonomers being provided in the first layer and the second half membranespanning monomers being provided in the second layer, the first halfmembrane spanning monomers being prevented from lateral diffusion in thefirst layer, the second half membrane spanning monomers being linked tothe membrane spanning lipids via the linker.
 6. A biosensor as claimedin claim 1 in which the ionophores are gramicidin or analogues thereof.7. A biosensor as claimed in claim 1 in which the enzyme to be detectedis a protease.
 8. A biosensor as claimed in claim 7 in which theprotease is Prostate Specific Antigen.
 9. A biosensor as claimed inclaim 1 in which the enzyme to be detected is a nuclease.
 10. Abiosensor for use in detecting the presence of an enzyme in a sample,the biosensor comprising a membrane and means for determining theimpedance of the membrane positioned relative to the membrane such thatthe impedance of the membrane may be determined, the membrane having aplurality of ionophores and a plurality of membrane-spanning componentstherein, the membrane-spanning components having attached thereto linkermolecules to which are connected the ionophores, the linker moleculesbeing cleavable by the enzyme to be detected, the cleavage of the linkermolecules causing a change in the ability of ions to pass through themembrane via the ionophores.
 11. A biosensor as claimed in claim 10 inwhich the membrane comprises a first and second layer of a closelypacked array of amphiphilic molecules and the membrane-spanningcomponents are prevented from lateral diffusion in the membrane.
 12. Abiosensor as claimed in claim 10 in which the ionophores comprise firstand second half membrane spanning monomers, the first half membranespanning monomers being provided in the first layer and the second halfmembrane spanning monomers being provided in the second layer with thefirst half membrane spanning monomers being prevented from lateraldiffusion in the first layer.
 13. A biosensor as claimed in claim 10 inwhich the ionophores are gramicidin or analogues thereof.
 14. Abiosensor as claimed in claim 10 in which the enzyme to be detected is aprotease.
 15. A biosensor as claimed in claim 14 in which the proteaseis Prostate Specific Antigen.
 16. A biosensor as claimed in claim 10 inwhich the enzyme to be detected is a protease.
 17. A biosensor for thedetection of enzymes comprising first and second zones, means to allowaddition of a sample suspected to contain a protease to the first zone,the first zone containing a probe linked to a carrier via a linkercleavable by the enzyme and means to allow passage of unlinked probefrom the first zone to the second zone; the second zone including amembrane the impedance of which is dependent on the presence or absenceof the probe and means to measure the impedance of the membranepositioned relative to the membrane such that the impedance of themembrane may be determined.
 18. A biosensor as claimed in claim 17 inwhich the membrane comprises a first and second layer of a closelypacked array of amphiphilic molecules and a plurality of ionophorescomprising first and second half membrane spanning monomers, the firsthalf membrane spanning monomers being provided in the first layer andthe second half membrane spanning monomers being provided in the secondlayer, the second half membrane spanning monomers being capable oflateral diffusion within the second layer independent of the first halfmembrane spanning monomers, the first half membrane spanning monomersbeing prevented from lateral diffusion in the first layer, and a ligandprovided on at least the second half membrane spanning monomers, saidligand being reactive with the probe or a portion thereof, the bindingof the probe to the ligand causing a change in the relationship betweenthe first half membrane spanning monomers and the second half membranespanning monomers such that the flow of ions across the membrane via theionophores is allowed or prevented.
 19. A biosensor as claimed in claim17 in which the enzymes to be detected are proteases.
 20. A biosensor asclaimed in claim 19 in which the protease is Prostate Specific Antigen.21. A biosensor as claimed in claim 17 in which the enzyme to bedetected is a nuclease.
 22. A biosensor as claimed in claim 17 in whichthe half membrane spanning monomers are gramicidin or analogues thereof.23. A biosensor as claimed in claim 17 in which the probe includes anionphore.
 24. A method of detecting the presence of an enzyme in asample comprising adding the sample to the biosensor as claimed in claim1 and measuring the change in impedance of the membrane.
 25. A method asclaimed in claim 24 in which the enzymes to be detected are proteases.26. A method as claimed in claim 25 in which the protease is ProstateSpecific Antigen.
 27. A biosensor for use in detecting the presence ofan enzyme or enzymes in a sample, the biosensor comprising a membraneand a detector positioned relative to the membrane for determining theimpedance of the membrane, the membrane having ionophores therein towhich are attached linkers, the linkers being cleavable by the enzyme orenzymes to be detected, the cleavage of a linker causing a change in theability of ions to pass through the membrane via the ionophores.